Methods for obtaining a genetically modified plant or microbe and for increasing oil yield

Abstract

Methods are provided for obtaining a genetically modified plant, wherein the plant exhibits an increased oil yield relative to a corresponding control plant that is not so genetically modified. The methods comprise genetically modifying a plant progenitor cell to cause a decrease in triose-phosphate isomerase activity and an increase in glycerol-3-phosphate dehydrogenase activity. The methods also comprise culturing the genetically modified plant progenitor cell to obtain the genetically modified plant. Also provided are methods for increasing oil yield, comprising genetically modifying a plant to cause, in at least one oil-producing organ or tissue of the plant, a decrease in triose-phosphate isomerase activity and an increase in glycerol-3-phosphate dehydrogenase activity. The genetic modification is carried out across more than a single generation. The genetically modified plant exhibits an increased oil yield relative to a corresponding control plant. Also provided are similar methods directed to a microbe.

1 . A method for obtaining a genetically modified microbe, wherein the microbe exhibits an increased oil yield relative to a corresponding control microbe that is not so genetically modified, comprising:
 genetically modifying a microbial cell to cause a decrease in triose-phosphate isomerase activity and an increase in glycerol-3-phosphate dehydrogenase activity; and   culturing the microbial cell to obtain the genetically modified microbe,   wherein the genetically modified microbe is selected from the group consisting of an oleaginous microbe, an oleaginous bacterium, an oleaginous actinomycetes, an oleaginous  Mycobacterium , an oleaginous  Streptomyces , an oleaginous  Rhodococcus , an oleaginous  Nocardia , an oleaginous fungus, an oleaginous yeast, and an oleaginous  Mortierella.      
     
     
         2 . The method of  claim 1 , wherein the genetic modification further causes at least one of an increase in malate dehydrogenase activity and an increase in ATP citrate lyase activity. 
     
     
         3 . The method of  claim 2 , wherein the genetic modification causes both the increase in malate dehydrogenase activity and the increase in ATP citrate lyase activity. 
     
     
         4 . The method of  claim 1 , wherein the genetic modification further causes at least one of a decrease in glyceraldehyde-3-phosphate dehydrogenase activity, an increase in fructose-1,6-bisphosphate aldolase activity, an increase in pyruvate kinase activity, and an increase in aconitase activity. 
     
     
         5 . The method of  claim 4 , wherein the genetic modification causes all four of the decrease in glyceraldehyde-3-phosphate dehydrogenase activity, the increase in fructose-1,6-bisphosphate aldolase activity, the increase in pyruvate kinase activity, and the increase in aconitase activity. 
     
     
         6 . The method of  claim 1 , wherein at least one of the decreases in activity and at least one of the increases in activity contribute to confer the increased oil yield of the genetically modified microbe. 
     
     
         7 . The method of  claim 1 , further comprising selecting the genetically modified microbe based on at least one of the decreases in activity and the increases in activity. 
     
     
         8 . The method of  claim 1 , wherein at least one of the decreases in activity is based on a technique selected from the group consisting of mutagenesis, RNAi, expression of siRNA, gene silencing, homologous recombination, disruption of a regulatory sequence, partial gene deletion, and full gene deletion. 
     
     
         9 . The method of  claim 1 , wherein at least one of the increases in activity is based on a technique selected from the group consisting of transformation, electroporation, transduction, introduction of recombinant DNA, introduction of a plasmid, and introduction of an artificial chromosome. 
     
     
         10 . The method of  claim 1 , wherein at least one of the decreases in activity is based on an effect selected from the group consisting of a decrease in specific activity of a corresponding enzyme, a decrease in copy number of a corresponding gene, a deleterious mutation in a corresponding gene, a deleterious modification of a corresponding enzyme, and a decrease in transcription of a corresponding gene. 
     
     
         11 . The method of  claim 1 , wherein at least one of the increases in activity is based on an effect selected from the group consisting of an increase in specific activity of a corresponding enzyme, an increase in copy number of a corresponding gene, an advantageous mutation in a corresponding gene, an advantageous modification of a corresponding enzyme, and an increase in transcription of a corresponding gene. 
     
     
         12 . The method of  claim 1 , wherein the oil yield of the genetically modified microbe is increased by at least 10% relative to the corresponding control microbe. 
     
     
         13 . A method of producing oil from a genetically modified microbe that exhibits an increased oil yield relative to a corresponding control microbe that is not so genetically modified comprising:
 obtaining the genetically modified microbe by the method of  claim 1 ; and   extracting oil from the genetically modified microbe.   
     
     
         14 . A method for increasing oil yield, comprising genetically modifying a microbe to cause a decrease in triose-phosphate isomerase activity and an increase in glycerol-3-phosphate dehydrogenase activity in the microbe, wherein:
 the genetic modification is carried out across more than a single generation of the microbe;   the genetically modified microbe exhibits an increased oil yield relative to a corresponding control microbe that is not so genetically modified,   and the genetically modified microbe is selected from the group consisting of an oleaginous microbe, an oleaginous bacterium, an oleaginous actinomycetes, an oleaginous  Mycobacterium , an oleaginous  Streptomyces , an oleaginous  Rhodococcus , an oleaginous  Nocardia , an oleaginous fungus, an oleaginous yeast, and an oleaginous  Mortierella.      
     
     
         15 . The method of  claim 14 , wherein the genetic modification further causes at least one of an increase in malate dehydrogenase activity and an increase in ATP citrate lyase activity. 
     
     
         16 . The method of  claim 15 , wherein the genetic modification causes both the increase in malate dehydrogenase activity and the increase in ATP citrate lyase activity. 
     
     
         17 . The method of  claim 14 , wherein the genetic modification further causes at least one of a decrease in glyceraldehyde-3-phosphate dehydrogenase activity, an increase in fructose-1,6-bisphosphate aldolase activity, an increase in pyruvate kinase activity, and an increase in aconitase activity. 
     
     
         18 . The method of  claim 17 , wherein the genetic modification causes all four of the decrease in glyceraldehyde-3-phosphate dehydrogenase activity, the increase in fructose-1,6-bisphosphate aldolase activity, the increase in pyruvate kinase activity, and the increase in aconitase activity. 
     
     
         19 . The method of  claim 14 , wherein at least one of the decreases in activity and at least one of the increases in activity contribute to confer the increased oil yield of the genetically modified microbe. 
     
     
         20 . The method of  claim 14 , further comprising selecting the genetically modified microbe based on at least one of the decreases in activity and the increases in activity. 
     
     
         21 . The method of  claim 14 , wherein at least one of the decreases in activity is based on a technique selected from the group consisting of mutagenesis, RNAi, expression of siRNA, gene silencing, homologous recombination, disruption of a regulatory sequence, partial gene deletion, and full gene deletion. 
     
     
         22 . The method of  claim 14 , wherein at least one of the increases in activity is based on a technique selected from the group consisting of transformation, electroporation, transduction, introduction of recombinant DNA, introduction of a plasmid, and introduction of an artificial chromosome. 
     
     
         23 . The method of  claim 14 , wherein at least one of the decreases in activity is based on an effect selected from the group consisting of a decrease in specific activity of a corresponding enzyme, a decrease in copy number of a corresponding gene, a deleterious mutation in a corresponding gene, a deleterious modification of a corresponding enzyme, and a decrease in transcription of a corresponding gene. 
     
     
         24 . The method of  claim 14 , wherein at least one of the increases in activity is based on an effect selected from the group consisting of an increase in specific activity of a corresponding enzyme, an increase in copy number of a corresponding gene, an advantageous mutation in a corresponding gene, an advantageous modification of a corresponding enzyme, and an increase in transcription of a corresponding gene. 
     
     
         25 . The method of  claim 14 , wherein the oil yield of the genetically modified microbe is increased by at least 10% relative to the corresponding control microbe. 
     
     
         26 . A method of producing oil, comprising:
 obtaining a genetically modified microbe by the method of  claim 14 ; and   extracting oil from the genetically modified microbe.